Cytochrome P-450 plays a key role in the metabolism, activation, and detoxification of drugs, endogenous steroids, xenobiotics, and environmental carcinogens. Cytochromes P-450 were purified from the livers of rats treated with phenobarbital (PB-P-450), 3-methylcholanthrene (MC-P-450), Beta-naphthoflavone (BNF-P-450), pregnenolone 16-Alpha-carbonitril (PCN-P-450), and the environmentally induced marine fish (Scup-P-450); monoclonal antibody-producing hybridomas were prepared to these enzymes. Among twelve hybrid clones producing MAbs active toward PB-P-450, seven were IgG1 and five were IgM. Three classes of MAbs were produced: one class bound but neither precipitated the PB-P-450 nor inhibited its aryl hydrocarbon hydroxylase (AHH) activity; a second class bound and immunoprecipitated, but did not inhibit enzyme activity; and a third class comprising a single clone, MAb 2-66-3, bound, immunoprecipitated and completely inhibited the AHH of PB-P-450. MAb 2-66-3 did not inhibit the AHH activities of MC-P-450, BNF-P-450, or PCN-P-450. The MAb 2-66-3 inhibited the AHH, ethoxycoumarin deethylase, and benzphetamine demethylase of liver microsomes from PB-treated rats, but did not inhibit these activities in microsomes from control, BNF-, or MC-treated rats. The MAb 2-66-3 showed high cross-reactivity in binding, immunoprecipitation, and inhibition of enzyme activity of PB-induced cytochrome P-450 from rabbit liver. Monoclonal antibodies to PCN-P-450 and Scup-P-450 are in the process of characterization. Monoclonal antibodies to different cytochromes P-450 will be extraordinarily useful for a variety of studies, including phenotyping, genetic analysis, and purification of cytochromes P-450.